Multilayered heparin hydrogel microwells for cultivation of primary hepatocytes

The biomaterial scaffolds for regenerative medicine need to be rationally designed to achieve the desired cell fate and function. This paper describes the development of hydrogel microstructures for cultivation of primary hepatocytes. Four different micropatterned surfaces are tested: 1) poly(ethyelene glycol) (PEG) microwells patterned on glass, 2) heparin hydrogel microwells patterned on glass, 3) PEG microwells patterned on heparin hydrogel-coated substrates, and 4) heparin hydrogel microwells patterned on heparin hydrogel-coated substrates. The latter surfaces are constructed by a combination of micromolding and microcontact printing techniques to create microwells with both walls and floor composed of heparin hydrogel. Individual microwell dimensions are 200 μm diameter and 20 μm in height. In all cases, the floor of the microwells is modified with collagen I to promote cell adhesion. Cultivation of hepatocytes followed by analysis of hepatic markers (urea production, albumin synthesis, and E-cadherin expression) reveals that the all-heparin gel microwells are most conducive to hepatic phenotype maintenance. For example, ELISA analysis shows 2.3 to 13.1 times higher levels of albumin production in all-heparin gel wells compared with other micropatterned surfaces. Importantly, hepatic phenotype expression can be further enhanced by culturing fibroblasts on the heparin gel walls of the microwells. In the future, multicomponent all-heparin gel microstructures may be employed in designing hepatic niche for liver-specific differentiation of stem cells.